Identification of Neoepitopes Recognized by Tumor-Infiltrating Lymphocytes (Tils) from Patients with Glioma

www.oncotarget.com Oncotarget, 2018, Vol. 9, (No. 28), pp: 19469-19480

Research Paper: Immunology Identification of neoepitopes recognized by tumor-infiltrating lymphocytes (TILs) from patients with glioma

Davide Valentini1, Martin Rao2, Qingda Meng2, Anna von Landenberg2, Jiri Bartek Jr3,4, Georges Sinclair4, Georgia Paraschoudi2, Elke Jäger5, Inti Harvey-Peredo4, Ernest Dodoo4 and Markus Maeurer1,2,5 1Centre for Allogeneic Stem Cell Transplantation (CAST), Karolinska University Hospital Huddinge, Stockholm, Sweden 2Therapeutic Immunology Unit (TIM), Department of Laboratory Medicine (LABMED), Karolinska Institutet, Stockholm, Sweden 3Department of Neurosurgery, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark 4Department of Neurosurgery, Karolinska University Hospital, Stockholm, Sweden 5 Krankenhaus Nordwest, Division of Oncology and Hematology, Frankfurt, Germany Correspondence to: Markus Maeurer, email: [email protected] Keywords: tumor-infiltrating lymphocytes; immunotherapy; neoepitopes; glioblastoma; interferon gamma; Immunology Received: September 11, 2017 Accepted: February 24, 2018 Published: April 13, 2018 Copyright: Valentini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

ABSTRACT

Neoepitope-specific T-cell responses have been shown to induce durable clinical responses in patients with advanced cancers. We explored the recognition patterns of tumor-infiltrating T lymphocytes (TILs) from patients with glioblastoma multiforme (GBM), the most fatal form of tumors of the central nervous system. Whole-genome sequencing was used for generating DNA sequences representing the entire spectrum of ‘private’ somatic mutations in GBM tumors from five patients, followed by 15-mer peptide prediction and subsequent peptide synthesis. For each mutated peptide sequence, the wildtype sequence was also synthesized and individually co-cultured with autologous GBM TILs, which had been expanded in vitro with a combination of interleukin (IL)-2, IL-15 and IL-21. After seven days of culture, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and/or IL-17A production was measured by ELISA in culture supernatants, and used as an epitope-specific immune response readout. Mutated peptides that induced a strong cytokine response were considered to contain legitimate neoepitopes. TILs from 5/5 patients with GBM exhibited specific immune reactivity profiles to the nominal target peptides, defined by IFN-γ and/or TNF-α production, as well as IL-17A. Neoepitopes, defined by mutated peptides inducing IFN-γ and/or TNF-α production without or only minimal reactivity to the wildtype sequences, were found for each individual patient. CD8+ TILs dominated the patients’ responses to private neoepitopes. The present study shows that neoepitope-specific TIL reactivity constitutes an important arm of anti-tumor immune responses in patients with GBM, and thus a powerful tool for developing next-generation personalized immunotherapies.

INTRODUCTION identified based on the amino acid sequence derived from antigens recognized by a specific population of T cells The identification of T-cell epitopes provided from defined by immune reactivity, i.e. cytokine production, host-derived targets is crucial in developing personalized usually measured as antigen-induced interferon gamma treatments for patients with advanced cancer. Epitopes are (IFN-γ) responses. This process does not only allow to www.oncotarget.com 19469 Oncotarget identify new cancer-specific targets, but also to identify way by which clinically relevant TIL responses can be T-cell receptors (TCR) that can be used in the development gauged and tailored for personalized immunotherapy. of immunotherapies. Several approaches of epitope identification are RESULTS currently in use. Mass spectrometry-based sequencing of peptides eluted from tumor cell-derived human leukocyte Clinical characteristics of patients antigen (HLA) molecules aims to decipher naturally processed and presented peptides, although there may Five patients were diagnosed with GBM (WHO be limits concerning sensitivity in this approach [1, 2]. grade IV tumor of the central nervous system), involving Screening of cDNA libraries encoding tumor-associated different anatomical compartments of the brain. A antigens (TAAs) has been used extensively; this approach description of the clinical characteristic of the patients is is labor-intensive and minimally effective, as larger provided in Table 1. transcripts and weakly expressed transcripts cannot be cloned easily. Furthermore, this method also eliminates TIL reactivity to wildtype and mutated peptides identification of some mutated antigens since it does not cover post-transcriptional modifications of protein A schematic representation of the entire process antigens [2]. The peptide-based screening approach workflow pertaining to the current study is presented identifies mutations through whole-exome sequencing in Figure 1. Immunoreactivity of the TILs from five followed by in-silico analysis, based on an algorithm different patients with GBM to private epitopes (patient- that predicts the peptide binding capacity of the major specific peptides generated from the whole-exome histocompatibility complex (MHC)-peptide complex [3, sequencing data) were evaluated in TIL cultures. A heat 4]. The drawback of this approach is that the antigen- map depicting cytokine responses (IFN-γ, TNF-α and/or receptor interaction is based on an algorithm that predicts IL-17A) following a 7-day in vitro stimulation assay with the MHC binding capacity, and is not thoroughly studied the wildtype as well as mutated peptide sequences was for MHC class II alleles or infrequent HLA alleles [2, 5]. generated for each patient (Figure 2). In Table 2, we show The discovery of epitopes using this approach is only as how many mutations from GBM lesions (5 patients) good as the in-silico prediction itself, which is limited are recognized by TILs based on IFN-γ production. for MHC class II alleles and for a number of MHC The amount of cytokine produced by TILs, considered class I molecules. A novel and revolutionary method as a readout for target-specific immunoreactivity, is the tandem minigene (TMG) approach, where the is presented based on a scale of low to high cytokine patients’ ‘private’ mutations are identified using whole- concentration in the culture supernatants (Figure 2). A exome sequencing in order to subsequently construct more comprehensive list, detailing individual cytokine a personalized library of gene sequences encoding concentration values (in pg/10e5 TIL/7 days) per peptide mutated epitopes – or neoepitopes. In the TMG setting, for the five patients with GBM is also presented in only a small portion of the gene around the mutation is Supplementary Table 1. Mutated and wildtype peptides synthesized, and its reaction with autologous T cells is alike were able to activate CD4+ and CD8+ TILs to tested to identify whether the predicted neoepitopes are produce a single cytokine or a combination of the three naturally processed and presented to the immune system cytokines measured (IFN-γ, TNF-α and/or IL-17A). [6] based on the assumption that the surrogate antigen- We noticed that a positive cytokine response by TILs presenting cells process and present the neoepitopes in a to the peptides tested (mutated and/or wildtype) was similar fashion to tumor cells. dominated by IFN-γ production. TILs from patients In the present study, we have used a combination of GBM-A and GBM-D appeared to exhibit the strongest whole-exome sequencing and individual peptide responses overall peptide-driven Th1 immune responses (IFN-γ to screen for immune reactivity among in vitro-expanded and/or TNF-α). Several of these peptides (mutated and/ tumor-infiltrating lymphocytes (TILs) from patients or wildtype) also resulted in IL-17A production by TILs. with glioblastoma (GBM) [7], the most lethal form of Patients GBM-A and GBM-B displayed only weak to brain cancer marked by a 5-year survival rate of under very low IL-17A responses to any of the peptides tested. 3% [8, 9]. The approach described here allows for direct While TILs from patient GBM-B produced loading of 15-mer peptides onto MHC class II molecules, measurable amounts of IFN-γ to twelve out of eighteen or by uptake, processing and subsequent presentation peptide pairs (mutated and/or wildtype) and did not mount of potential epitopes by MHC class I or MHC class II a strong IL-17A response to any of the peptides tested, molecules on surrogate antigen-presenting cells (APCs) TILs from patient GBM-E produced substantial amounts to enable T-cell engagement and activation. We report of IFN-γ in response to ten out of eleven mutated peptides. a method that helps the identification of antigenic (neo) Interestingly, TILs from patient GBM-E also produced a epitopes that are presented either directly or indirectly (via combination of IFN-γ and IL-17A in response to three processing) to T cells in the tumor microenvironment, a mutated peptides. In terms of peptide-driven TNF-α www.oncotarget.com 19470 Oncotarget Table 1: Clinical characteristics of patients Patient ID Age Sex Diagnosis Grade Tumor localization GBM-A 67 M GBM IV Thalamus GBM-B 62 F GBM IV Temporal GBM-C 63 F GBM IV Temporal GBM-D 65 M GBM IV Frontal GBM-E 67 F GBM IV Parietal

Figure 1: Schematic representation of process workflow. Tumor tissue and autologous skin biopsies from patients with GBM (n = 5) were submitted for genomic DNA extraction and whole-exome sequencing. The list of private mutations unique to each patient, alongside the corresponding native nucleotide sequence were translated into peptide sequences. 15-mer peptide sequences, with the mutated amino acid in the middle (thus flanked by 7 amino acids on either side) were synthesized. The resulting mutated (mut) peptides as well as the wildtype (wt) counterparts were exposed to autologous tumor-infiltrating lymphocytes over a 7-day period to induce cytokine production, which was then measured using commercially available ELISA kits. www.oncotarget.com 19471 Oncotarget Figure 2: Cytokine production by TILs in response to predicted mutated peptides and corresponding wildtype sequences. TILs from five patients with GBM were exposed to mutated peptides predicted to be private tumor-specific epitopes, along with the corresponding wildtype sequences. After seven days of culture, supernatants were harvested for measurement of IFN-γ, TNF-α and IL-17A by sandwich ELISA. Based on the actual concentration of cytokines produced by TILs co-cultured with the individual wildtype or mutated peptides, heat maps were created depicting the peptide-induced responses unique to each patient. A scale ranging from low (black) to high (red) amounts of cytokine produced by total TILs, the CD4+ as well as the CD8+ TIL subsets are shown. Also shown in the scale are cytokine concentration values ranging from 0–4804 pg/10e5 TIL/days. The identical gene name appears several times for some patients due to recognition of different epitopes/peptides belonging to the same gene product. Actual cytokine concentrations are provided in Supplementary Table 1. www.oncotarget.com 19472 Oncotarget Table 2: Number of mutations that were recognized by TILs/number of mutations identified by whole DNA exome sequencing GBM-A GBM-B GBM-C GBM-D GBM-E TILs 16/48 17/26 4/92 16/75 9/22 CD8+TILs 6/48 14/26 4/92 10/75 7/22 CD4+TILs 4/48 16/26 0/92 5/75 6/22

production by TILs, positive responses were seen mostly to a mutated peptide and its corresponding wildtype with wildtype peptides, with some exceptions (GBM-A counterpart from the preferentially expressed antigen = two mutated epitopes, GBM-C = one mutated epitope, in melanoma family member 4 (PRAMEF4) protein, GBM-D = four mutated epitope). TILs from patient which belongs to a group of previously described human GBM-C had the weakest IFN-γ response to the peptides melanoma-specific cytotoxic T-lymphocyte antigens with tested, but also the strongest TNF-α response to the a potential role in retinoic acid signaling [12]. Similarly, wildtype peptides, compared to all five patients. TIL from TILs from patient GBM-D produced high amounts of this patient also showed a measurable IL-17A response, IFN-γ in reaction to both a wildtype and mutated peptide particularly to wildtype peptides. Five mutated peptides pair derived from the ankyrin repeat domain-containing induced only IL-17A production in TILs from three protein 36C (ANKRD36C), which is expressed on patients (GBM-A = two peptides; GBM-C = two peptides; immune cells (including lymphocytes) with ion channel- GBM-D = one peptide), albeit in the low to medium range inhibitory properties. Strong IFN-γ production in response (median concentration value = 138.6 pg/ml). None of to a wildtype peptide of the stem cell factor or Kit, which the peptides tested (mutated and/or wildtype) induced a is also implicated in the regulation of antigen-specific combination of TNF-α and IL-17A production in lieu of T-cell memory [13, 14], was also seen among TILs from IFN-γ by TILs from any of the patients with GBM. patients GBM-A. When the individual peptides were tested for TILs from patient GBM-C showed a strong IL-17A activation of (sorted) CD4+ or CD8+ TIL subsets in regard response to a wildtype peptide sequence derived from to IFN-γ production, there appeared to be a generally epidermal growth factor receptor (EGFR), and a strong stronger CD8+ rather than CD4+ TIL response to the test TNF-α response to a wildtype survivin (BIRC5) peptide. candidate peptides from 5/5 patients with GBM (Figure Pertaining to immune responses to proteins associated 2/Table 2). CD8+ TILs from patient GBM-A exhibited a with the central nervous system, we identified IFN-γ broader IFN-γ production in response to wildtype peptides and TNF-α responses to a mutated peptide of teneurin (twelve peptides) as compared to the mutated sequences transmembrane protein 3 (TENM3), which is involved in (six peptides). CD4+ TILs from the same patient had a neuronal and potentially visual development [15] (TILs similar response pattern to wildtype (six peptides) as well from patient GBM-D). TILs from patients GBM-A and as mutated peptide sequences (four peptides). Patient GBM-B had measurable IFN-γ and/or TNF-α reactivity to GBM-B also displayed strong CD8+ and CD4+ TIL wildtype as well as mutated peptides from the homeobox responses to wildtype peptides, but an equally potent protein Hox-A1 (HOXA1), which plays an important role production of IFN-γ was also observed with the mutated in hindbrain and cardiovascular development [16]. TILs sequences. Patient GBM-C only showed IFN-γ responses from patient GBM-C had measurable IFN-γ responses to by CD8+ TILs to two wildtype peptides and four mutated a mutant peptide of the ATP binding cassette subfamily A peptides – which appeared to be more focused. While member 2 (ABCA2), a protein that is strongly expressed in CD8+ TILs from patient GBM-D had comparable the brain, with involvement in neuronal development [17]. responses to the wildtype and mutated peptide sequences, Some striking TIL responses to peptides related the CD4+ TILs recognized five mutated peptides and none to the immune system were also observed. TILs from of the wildtype sequences. Last but not least, TILs from patients GBM-A and GBM-D showed immune reactivity patient GBM-E had both CD8+ and CD4+ TILs with a (IFN-γ and TNF-α production, respectively) to a wildtype rather highly focused IFN-γ response pattern, recognizing peptide from interferon regulatory factor 5 (IRF5), a mainly mutated sequences rather than the wildtype transcription factor that is involved in the functionality peptides. and phenotypic polarization of macrophages [18]. Strong We also examined in greater detail genes encoding IFN-γ response to a wildtype as well as mutated peptide the protein source of the respective peptides tested, in sequence derived from apoptotic inhibitor 5 (API5) were relation to the cytokine responses. The Gene Cards online observed in TIL from patient GBM-A. The abrogation of suite (www.genecards.com) [10] and the UniProt database API5 protein expression in human cervical cancer cells (www.uniprot.org) [11] were used as a reference. TILs has been implicated in tumor regression and sensitivity from patient GBM-A showed very strong IFN-γ responses to chemotherapy [19]. Patient GBM-C had TILs which www.oncotarget.com 19473 Oncotarget responded with TNF-α production to a mutated peptide and presentation by the MHC class I – class II pathways. The provided by the transforming growth factor beta possibility to revitalize and restore in a robust fashion the regulator 4 (TBRG4), which is involved in cell growth neoantigen-specific immune reactivity of TIL populations in and proliferation via the transforming growth factor vitro with IL-2, IL-15 and IL-21 conditioning holds promise beta (TGF-β) signaling pathway. A recent study showed for future clinical protocols in immuno-oncology. The use of that gene silencing of TBRG4 using short-hairpin RNA the TMG method (described in the introductory section) will structures reduces the viability of the U87MG primary aid the study of whether wildtype and/or mutant epitopes can human GBM cell line in culture [20]. In addition, CD8+ be naturally processed and presented to T cells (and TILs in TILs also produced IFN-γ in response to a mutated peptide particular), although these experiments are usually performed derived from leukocyte immunoglobulin like receptor using B cells or dendritic cells from the respective patient. B3 (LILRB3), which has a role in reducing non-specific Furthermore, the presentation of the nominal target antigen inflammation, upregulating the MHC-I pathway and (thus, epitopes) by MHC molecules on transformed cells may focusing the cellular immune response [21]. In a control be substantially different [23, 24]. experiment, we also observed IFN-γ responses to mutated It is important to note that some mutated peptides peptides derived from TBRG4, LILRB3 as well as EGFR derived from GBM tumors are exclusively recognized, yet among peripheral blood T cells from patient GBM-C, in not their wildtype counterparts. Other patterns of immune addition to some unique recognition patterns compared to reactivity show stronger recognition of the mutated epitope TILs (Supplementary Table 2). compared to the wildtype peptide sequence. Positive Mutated peptides that induced an IFN-γ and/ cytokine responses i.e. IFN-γ/TNF-α production, in or TNF-α response (exclusively or stronger than the response to stimulation with mutated peptide sequences corresponding wildtype peptide) were selected and listed indicate that T-cell populations are present within TIL that in a table (Table 3). This list also includes the mutated have the capacity to recognize mutant target epitopes if peptides that induced IL-17 production, in addition to exclusively the mutant target epitope is recognized (and IFN-γ and/or TNF-α. not the corresponding wildtype epitope), it is highly likely that TIL have been expanded and stimulated by naturally DISCUSSION presented T-cell neoepitopes Such uniquely recognized mutated epitopes may present excellent candidates for The present study describes T-cell epitope mapping additional selection and expansion of tumor-specific that combines whole-exome sequencing, identification of T cells, in particular TILs directed against neoepitopes private mutations in a patient’s tumor DNA (compared that are preferably or exclusively generated by mutational to non-transformed tissue), synthesis of the respective events (mutations, splicing or stop codons) [25]. This is mutated peptide sequence as well as the corresponding particularly important for GBM, given the high degree of wildtype sequence for comparison and finally testing genetic heterogeneity within the tumor before and after T-cell responses directed against specific mutated and chemotherapy [26, 27]. However, T-cell responses within wildtype epitopes found within the peptide sequences. TIL are usually polyclonal and the ‘net-response’ of The detection of an immune response to neoepitopes cytokine production may therefore stem from several T-cell among cytokine-expanded TILs, in particular CD8+ clones directed against the same nominal target. A detailed TILs may reflect (i) existence of T cells in the tumor examination of TIL reactivity of mutant versus wildtype microenvironment harboring neoepitope-specific TCRs; epitope sequences would therefore require T-cell cloning (ii) neoepitopes that induce a positive T-cell response are and a more detailed molecular composition analysis of TIL in fact naturally presented to the immune system by the populations directed against defined tumor epitopes. corresponding HLA alleles and do not represent simply an Anti-tumor responses are in effect productive artefact; (iii) potential cross-reactivity of neoepitopes with and very focused autoimmune responses that lead to TCRs of other specificities due to molecular mimicry or beneficial clinical outcomes. In this regard, some TIL epitope spreading. It is also possible that naturally processed subpopulations (in particular neoepitope-specific CD8+ epitopes within the tumor could be aberrantly processed T cells) may be polyfunctional, since they are capable of or trimmed by the endoplasmic reticulum aminopeptidase cytokine production as well as cytotoxic activity [25, 28]. 1 (ERAP1) protein within the endoplasmic reticulum The nature of the epitope itself, its capacity to bind to (ER), leading to degenerate, non-immunogenic MHC-I- the groove of the MHC molecule and the strength of compatible sequences [22]. Induction of productive anti- recognition by the TCR (affinity) are collectively reflected tumor CD8+ TIL responses may therefore be subdued in in the cytokine release assays reported here. Production of the tumor microenvironment and mutant epitopes may not pro-inflammatory cytokines such as TNF-α, IFN-γ, type bee seen by the cellular immune system, due to differential 1 interferons (IFN-α/β) and IL-18 may increase antigen processing and presentation by MHC molecules. Vice versa, turnover and promote further activation of a wider range of wildtype epitopes may also not be presented and the (point) bystander immune effector cells [29, 30]. Thus, an optimal mutation itself may render the epitope fitting for processing immune response elicited by only a small population of www.oncotarget.com 19474 Oncotarget Table 3: List of mutated peptides with promising neoepitope characteristics Patient IFN-γ TNF-α IFN-γ + TNF-α ID Sequence Gene ID Sequence Gene ID Sequence Gene ID GBM-A QVLLEGEHCWLGAKV PAPPA2 HPLPSAEQYIDFCES CRHBP HRKSLLLTIFQWKF NF1 ILLSLGF. ATP8B3 HHHHHHHRHPQPATY HOXA1 GBM-B RVLFVVFIYVVTVCG OR4C3 LVVGRFVSLRTEIKP FAM20B APLSAALKSLKRAAG INSM2 IREQEEMTQEQEEKM GOLGA6L22 PTGDLFSILDFPFLY TMEM241 DIKEKLCFVALDFEQ POTEJ MSQSRHRL PRKRA VILFRLLVVILFGRL C9orf57 GBM-C SNPHLLSLPSEPLEL LILRB3 WSIGVICSILVSGLS MYLK TLVKRPAKPGGPQEP ABCA2 GBM-D PDAVGKCGSAGIKVI ATP1A2 ADPIPSGRSPGPCGA LINC00273 GKGVMLAISQGRVQT TENM3 EGGPAAPHLGSRTAP LINC00273 NRPTSGPRQRHTRRS LINC00273 DMYGTGQQSLYS CDH7 QSYKNDFSAEYSEYR ELL ARKAKYNLHATVRYQ NCAN FARKLKDIHETLGFP TTN EPDNIKYMISEEKGS TTN AATSHPKHIKPATSH GPR50 GBM-E SSGGCCGSSSGGCCS LCE1F REDAGAGEEDVGAGG GOLGA6L1 IREQEEMIREQEAQR GOLGA6L2 PPTWSGRHAPGDRDN LOC645752 QFLIPTSFSVSSNSV DSPP Mutant peptides that induced strong IFN-γ and/or TNF-α responses, including those eliciting a measurable IL-17A response were listed for each patient. Also shown are proteins from which the respective peptide sequences are derived. The gene IDs LINC00273 and TTN appear twice for patient GBM-D due to recognition of different epitopes/peptides derived from the same protein products. The point mutation within the peptide sequence is marked in red (please refer to Supplementary Table 1 for the corresponding wildtype sequences).

neoepitope-specific TILs may lead to the mobilization of clinically beneficial effects in patients with metastatic other T cells as well as B cells – which may potentially be cancer. Patients with GBM, in particular those who have reflected in neoepitope-specific antibody responses. recurrent disease, are likely to develop further mutations TILs have also been proposed as a reliable biomarker in the tumor after temozolomide therapy [27]. Thus, a of the efficacy of pharmacological agents, as shown in the mutational analysis of the tumor following chemotherapy example of breast cancer treatment [31, 32]. In addition, may uncover neoepitopes, which could elicit T-cell neoepitope-specific T cells have been identified as an reactivity and be used for adjunct immunotherapy. The important target of immune checkpoint blockade therapy of delivery of tumor-reactive T cells could either be systemic, patients with metastatic melanoma or non-small cell lung or intra-cranial, as reported using chimeric antigen receptor cancer i.e. treatment with anti-programmed cell death-1 (CAR) transgenic T cells to patients with GBM [34, 35], the (PD-1) and/or anti-cytotoxic T lymphocyte-associated same route of delivery maybe considered for neoepitope- antigen-4 (CTLA-4) monoclonal antibodies [33]. A more specific T cells after surgery [36]. clonal neoepitope response, hence a stricter TCR repertoire We noticed that patients GBM-C (770 days) and of T cells, appears to promise a higher likelihood of eliciting GBM-E (582 days) had a generally better survival profile www.oncotarget.com 19475 Oncotarget post-surgery compared to the other three patients (between TIL responses are mapped for IFN-γ, as well as for IL-17 300 and 550 days). TIL from both patients exhibited production. Neoepitope-induced immunological tolerance, focused IFN-γ responses to neoepitopes; patient GBM-C owing to enhanced regulatory T-cell (Treg) activity can had an exclusively CD8+ TIL-centric reactivity while be a limiting factor in clinical efficacy after reinfusion of patient GBM-E exhibited a combination of CD4+ and TILs [44]. This effect may however by counteracted by CD8+ TIL responses to neoepitopes. In addition, TILs from the presence of IL-21 in the T-cell expansion medium, patient GBM-E recognized mutated peptides involved in the since effector CD4+ and CD8+ T cells have been shown TGF-β signaling pathway, which has a poor prognosis in to develop resistance to CD4+CD25+ Treg-mediated human GBM [20, 27], the ABCA2 cellular protein that is immune suppression following exposure to IL-21 [45]. highly expressed in the brain (recognized by CD8+ TILs), We have also previously shown that GBM TILs cultured as well as LILRB3, which is implicated in fine-tuning the with IL-2, IL-15 and IL-21 have very low frequencies of MHC-I-dependent immune response (recognized by CD8+ FoxP3+ CD4+ CD25hi Tregs after the expansion phase TILs) [21]. This observation suggests a possible correlation [7]. The fact that the expansion protocol used in this study between focused neoepitope-specific T-cell responses and does not induce Tregs is highly desirable for clinical use, a survival benefit among patients with GBM, as previously since the presence of Tregs in the infusion product may reported for patients with metastatic melanoma or non- dampen the expected anti-tumor effect of TILs in the small cell lung cancer [33]. patients. TIL infusion products with very low or no Tregs The existence of measurable and potentially durable therefore represent enhanced quality with greater chances immune responses directed against a distinct repertoire of of mediating productive immune responses and positive private neoantigens in patients with advanced cancers has clinical outcomes. clinically relevant implications also for the development In conclusion, neoepitope identification using the of personalized cancer vaccines [37]. Conjugate vaccine method described in this study allows the characterization constructs encompassing various patient-specific of TIL populations that are responsive to autologous neoepitopes can be formulated for delivery alongside a cancer cells based on cytokine production (IFN-γ alone potent adjuvant [37, 38]. Vaccines may then be administered or in combination with other cytokines, e.g. IL-17). systemically, or via the intra-arterial as well as intra-cranial Furthermore, by combining the readout obtained by (intra-tumoral) routes, depending on feasibility of surgical peptide-based screening and the TMG approach, it intervention and the vaccine formulation, particularly since is possible to narrow down the epitope repertoire for peripheral blood mononuclear cells (PBMCs) are also able selective and specific design of TCRs for developing to react (without ex vivo stimulation) to mutant epitopes, efficacious personalized treatments for patients with that are either unique or shared with neoepitopes recognized advanced cancers. The fast screening approach will also by TILs (Supplementary Table 2). allow for the better selection of TILs to be reinfused into We also measured IL-17A TIL responses in this the patient as adjunctive immunotherapy. study, although the role of this pro-inflammatory cytokine in cancer has been controversial. Studies have shown MATERIALS AND METHODS that IL-17 may have a beneficial effect on tumor growth and disease progression, while some preclinical research Patient characteristics efforts indicate that IFN-γ-dependent IL-17 production by T cells can lead to elimination of tumors in vivo and GBM tumor samples (n = five patients; designated establish tumor-specific immune responses [39, 40]. as GBM-A to GBM-E) were used to generate TILs, Furthermore, IL-17 has also been attributed to induction which was approved by the Regional Ethical Review of tumor-specific cytotoxic T-lymphocytes in the tumor Board (Regionala etikprövningsnämnden) at Karolinska microenvironment, in part due to increased MHC class I Institutet, Sweden (EPN: 2013/576-31 & 2013/977-31/1). and II antigen processing and presentation [41, 42]. In a A brief description of the patients’ clinical characteristics comprehensive review by Punt et al., a higher frequency is provided in Table 1. of IL-17+ T cells in tumor tissue as well as peripheral blood from patients with various types of solid cancers Generation of TILs from GBM tumors appeared to correlate with improved survival [43]. This evidence therefore prompted us to include IL-17-inducing TILs from five patients with GBM were isolated and mutated peptides in the selection of potential neoepitopes propagated in vitro (in the presence of IL-2, IL-15 and IL-21) associated with GBM, with the condition that IFN-γ as previously described [7, 46]. Briefly, tumor tissue was cut responses were also observed. It is therefore plausible into 1–2 mm3 pieces with a sterile scalpel, washed twice with that selective IL-17 responses to exclusively mutated ice-cold PBS and cultured in 24-well plates in T-cell medium but not the wildtype epitopes may give rise to clinically (Cellgro GMP-grade serum-free medium; CellGenix, beneficial responses i.e. tumor regression. This can be Catalogue Number: 20801-0500) with 5% pooled human tested in future clinical trials, where mutation – specific AB serum (Innovative Research, Catalogue Number: IPLA- www.oncotarget.com 19476 Oncotarget SERAB-14668), supplemented with recombinant human (rh) spliced products or stop codons may result in epitopes that are IL-2 (1000IU/ml) (Prospec, Catalogue Number: cyt-209-b), shorter than the 15-mer standard peptides used for screening rhIL-15 (10 ng/ml) (Prospec, Catalogue Number: cyt-230-b) of immune cell reactivity and were therefore omitted. and rhIL-21 (10 ng/ml) (Prospec, Catalogue Number: cyt- 408-b). Medium was changed as necessary. Feeder cells, Neoepitope synthesis and immune reactivity consisting of allogeneic PBMCs which were irradiated at 55 evaluation in TILs Gy, were added at a ratio of 1 (feeder cells):10 (TILs) on day 7 of culture. Further expansion of the TILs was performed in After identification of mutations through WES and G-Rex flasks (Wilson Wolf, Catalogue Number: 800400S) in-silico analysis of the sequencing data, 15-mer peptides with 30 ng OKT3/mL (Miltenyi Biotec, Cat #: 170-076-124) were constructed by placing the mutation at the center and irradiated allogeneic feeder cells at one feeder cell per of the 15-amino acid sequence (customized order from five TILs. Peptide & Elephants, Catalogue Number: A15-554LB). The corresponding wildtype epitope to each mutated DNA isolation, whole-genome sequencing and sequence was also synthesized for comparison of immune 5 mutanome analysis cell reactivity. TILs (1.0 × 10 cells) from patients with GBM were cultured in 200 μl of T-cell medium with Genomic DNA purification, library construction, 1 μg/ml of the individual wildtype or mutated peptide exome capture of approximately 20,000 coding genes, in round-bottom 96-well microtiter plates. Negative and next generation whole-exome sequencing (WES) controls contained assay medium alone while the anti- of tumor and normal samples isolated from PBMCs human CD3 antibody (OKT3, 30 ng/mL) was used as the were performed using standard operating procedures as positive control of maximal TCR stimulation. Cells were previously described by Jones et al. [47]. In brief, genomic incubated for 7 days in a CO2 incubator at 37° C, after DNA from tumor and normal samples were fragmented, which supernatants were harvested and tested for IFN-γ purified and used for constructing an Illumina DNA production using a standard sandwich enzyme-linked library (original method by Illumina, San Diego, CA, immunosorbent assay (ELISA) kit (Mabtech, Stockholm, USA; modifications described in detail by Jones et al. Sweden). Values from the negative control (medium) were [47]). Exonic regions were captured in solution using the subtracted from antigen (peptide)-specific responses and the data reported to reflect the IFN-γ production (in pg/7 Agilent SureSelect Human All Exon 50 Mb kit Version 5 3 according to the manufacturer’s instructions (Agilent, days/1.0 × 10 TILs representing the net IFN-γ production Catalogue number: G3370A). Paired-end sequencing, from CD4+ or CD8+ T-cell populations. Cultures of CD4+ and CD8+ TILs with the individual peptides were resulting in 100 bases from each end of the fragments, performed after separation of the T-cell subsets from total was performed using a HiSeq 2000 Genome Analyzer TIL cultures using the MACS anti-human CD4+ (Miltenyi (Illumina, Catalogue number: SY-401-1001). Sequence Biotec, Catalogue Number: 130-045-101) and anti-human data were mapped to the reference human genome CD8+ (Miltenyi Biotec, Catalogue Number: 130-045-201) sequence and sequence alterations were determined by microbeads as well as elution columns (Miltenyi Biotec, comparison of over 50 million bases of tumor and DNA Catalogue Number:130-042-401). from non-malignant lesions. Bases of sequence data were obtained for each sample, with a high fraction from the Isolation of and immune reactivity evaluation in captured coding regions. Over 43 million bases of target DNA were analyzed in the tumor and normal samples, PBMCs and an average of 42 to 51 reads were obtained at each PBMCs were isolated from heparinized blood or base in the normal and tumor DNA samples. The tags blood products collected from apheresis process over a were aligned to the human genome reference sequence ficoll hypaque gradient. Similar to the in vitro stimulation (hg18) using the Eland algorithm of CASAVA 1.6 software of TILs, (unseparated) PBMCs (1.0 × 104/well) from (Illumina, San Diego, CA, USA). The chastity filter of the patients GBM-C and GBM-E were co-cultured with the BaseCall software of Illumina was used to select sequence wildtype and mutated peptides for seven days at 37° C and reads for subsequent analysis. The ELANDv2 algorithm of with 5% CO2. Cell culture supernatants were harvested at CASAVA 1.6 software (Illumina, San Diego, CA, USA) the end of the incubation period for IFN-γ detection by was then a applied to identify point mutations and small sandwich ELISA. insertions, deletions or stop codons. Mutation polymorphisms recorded in the Single Nucleotide Polymorphism Database ELISA for cytokine detection (dbSNP) were removed from the analysis. Potential somatic mutations were filtered as previously described [47]. Only ELISA was used for detecting the production of non-synonymous single and dinucleotide substitutions, IFN-γ (Mabtech, Catalogue Number: 3420-1H-20), TNF-α respectively were listed in an Excel spreadsheet. Alternatively (Mabtech, Catalogue Number: 3510-1H-20) or IL-17A www.oncotarget.com 19477 Oncotarget (Mabtech, Catalogue Number: 3520-1H-20) in cell culture Slingluff CL Jr, Ross MM. The peptide recognized by HLA- supernatants according to the manufacturer’s instructions. 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